Vitamin D Metabolite Analysis in Biological Samples Using Agilent Captiva EMR—Lipid

Importance of the Topic

Vitamin D status is commonly assessed by measuring 25-hydroxyvitamin D2 and D3 in plasma or serum. Accurate quantification is essential for evaluating nutritional status, guiding supplementation, and supporting clinical research. However, lipids and phospholipids in biological samples compromise sensitivity and reproducibility in LC/MS/MS assays. An efficient sample cleanup that removes lipid interferences without affecting target analytes is critical to deliver reliable analytical results.

Study Objectives and Overview

This application note evaluates a novel in situ protein precipitation and lipid-removal workflow using Agilent Captiva EMR–Lipid on a 96-well format. The study aimed to verify accuracy, precision, and matrix cleanup for 25-OH D2 and D3 in human serum across multiple days and QC levels. Key performance metrics included intra- and interday reproducibility, absolute recovery, and phospholipid removal efficiency.

Methods and Instrumentation

Sample preparation combined protein precipitation with pass-through lipid cleanup using Captiva EMR–Lipid sorbent in a 96-well plate format. Serum samples were mixed with acidified acetonitrile, precipitated, and then drawn through the EMR–Lipid sorbent under controlled vacuum to capture lipids while allowing target metabolites to elute.

• LC Configuration:
   Agilent 1290 Infinity II pump, multisampler, multicolumn thermostat
   InfinityLab Poroshell 120 EC-C18 analytical column (2.1×50 mm, 2.7 µm) with guard column
   Mobile phases: water + 0.1 % formic acid (A), methanol + 0.1 % formic acid (B); flow 0.5 mL/min; 75–98 % B gradient
• MS/MS Configuration:
   Agilent 6460 Triple Quadrupole with Jet Stream ESI in positive mode; dynamic MRM
   Drying gas (250 °C), sheath gas (325 °C), capillary 5000 V; optimized collision energies for each transition

Key Results and Discussion

Calibration curves covering 10–750 ng/mL for D2 and 20–750 ng/mL for D3 exhibited R2 > 0.992. Intra- and interday accuracy at all QC levels ranged from 90 % to 110 %, with precision < 10 % RSD. Absolute recoveries averaged 89–106 % (RSD < 15 %), confirming no retention of vitamin D metabolites by the sorbent. Phospholipid removal exceeded 99.5 %, as shown by precursor ion scans at m/z 184. Captiva EMR–Lipid cleanup delivered consistent peak areas (%RSD < 3 %), whereas protein precipitation alone showed up to 80 % signal suppression and > 25 % variability. Postcolumn infusion confirmed elimination of coeluting suppression zones, enhancing analytical sensitivity.

Benefits and Practical Applications

• Streamlined workflow: single-step protein crash and lipid removal in one plate
• Enhanced robustness: minimal carryover, no clogging, reproducible vacuum-controlled flow
• Improved sensitivity: reduced matrix effects and suppression lead to sharper peaks and lower detection limits
• Scalability: 96-well format suitable for high-throughput clinical and research laboratories

Future Trends and Opportunities

The selective EMR–Lipid sorbent chemistry can be extended to multiclass drug screening and food safety applications where lipid interference is pervasive. Future work may integrate this cleanup strategy with online extraction, automation platforms, or broaden its use in challenging matrices such as tissue homogenates and complex food extracts. Combining EMR–Lipid with advanced mass spectrometers could further lower quantitation limits and support large-scale population studies.

Conclusion

Agilent Captiva EMR–Lipid provides a rapid, reliable solution for simultaneous protein precipitation and lipid removal in vitamin D metabolite analysis. The method demonstrates excellent accuracy, precision, and matrix cleanup, substantially improving assay robustness and sensitivity compared to conventional precipitation. Its ease of use and high throughput capability position it as a valuable tool for clinical research and routine laboratory testing.

References

1. L. Zhao, D. Lucas, Agilent Technologies, publication number 5991-8006EN.
2. U.S. FDA, HHS, Center for Drug Evaluation and Research, Guidance for Industry Bioanalytical Method Validation, 2001.
3. L. Zhao, D. Lucas, Agilent Technologies, publication number 5991-8007EN.